Our program aims to elucidate fundamental principles of protein folding in the formation of voltage-gated potassium (Kv) channels, including events from inception in the ribosomal tunnel to integration into the ER bilayer. Knowledge of the mature Kv channel in the plasma membrane cannot tell us about the channel's history of distinct biogenic stages; thus we focus on early folding events. We use novel applications of our recently developed folding and accessibility assays, new unnatural amino acids/synthetase inhibitors, real-time translation kinetics, electrophysiology of Xenopus oocytes, photocrosslinking in the ribosomal tunnel and translocon, and computational approaches to probe the three major domains of a Kv channel (T1, voltage- sensor, and pore) and define peptide-tunnel interactions governing protein folding and elongation. Five interrelated Projects, with 11 Aims, comprise this grant proposal. First, we will determine if folding of T1 subdomains are coupled or independent events, and establish the role of the T1-S1 linker in intersubunit tetramerization. These studies will define crosstalk between T1 subdomains and T1's flanking sequences, providing a previously unknown molecular basis for T1 formation. Second, we will elucidate key folding events in the biogenesis of a voltage-sensor, namely, i) the conformation of S3 and S4 as they move through the translocon into the ER membrane, a critical stage for correct insertion into the ER membrane, and ii) the principles underlying helix formation in the biogenically unique S2 segment. Third, we will define mechanisms governing formation of the permeation pore, specifically the biogenic role of early re-entry of the pore helix and its consequences for channelopathy. We will test whether the re-entrant pore helix orients S5 and S6 for folding events and identify residues that have co-evolved for pore formation. Fourth, we identify `speed bumps' in the ribosomal tunnel that modulate peptide movement and key sensing/signaling zones that induce peptide rearrangements during protein synthesis. To this end, we are developing new technologies to assess peptide transit in real time, a factor that modulates correct peptide folding. Finally, the last Project will test whether peptides traverse idiosyncratic tunnel routes, whether electrostatic screening in the tunnel contributes to propagated peptide rearrangements, and whether a peptide's C-terminal secondary structure in the tunnel can be reconfigured by the folded state of its N-terminus outside. These five Projects will 1) establish new paradigms for peptide-ribosome interactions that govern protein folding, and 2) define Kv channel formation. Both generalize to folding mechanisms for all proteins to avert disease.